Recombinant Peptide Fusion Protein-Templated Palladium Nanoparticles for Suzuki-Miyaura and Stille Coupling Reactions

Article abstract of DOI:10.1002/cctc.201902099

This study examined the use of nanoparticles created with recombinant 45-amino acid long peptides fused to green fluorescent protein (GFPuv) to catalyze twelve representative Suzuki-Miyaura and Stille coupling reactions. A method was developed to prepare powders (Pd@GFP) containing protein and synthesized nanoparticles. Next, coupling reactions were performed in a green solvent without nanoparticle purification. Pd@GFP had high turnover frequencies for the synthesis of model compounds including lapatinib (Tykerb) and could be recycled. This study establishes a potentially cost-effective approach to prepare heterogeneous catalysts containing well-defined nanoparticles enabling key C?C bond formation leading to synthetically and pharmaceutically interesting compounds.

Full text of DOI:10.1002/cctc.201902099

Accepted Article
Title: Recombinant Peptide Fusion Protein-templated Palladium
Nanoparticles for Suzuki-Miyaura and Stille Coupling Reactions
Authors: Imann Mosleh, Hamid R. Shahsavari, Robert Beitle, and M.
Hassan Beyzavi
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ChemCatChem
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COMMUNICATION
Recombinant Peptide Fusion Protein-templated Palladium
Nanoparticles for Suzuki-Miyaura and Stille Coupling Reactions
[
a]
[b,c]‖
[a]
[b]
Imann Mosleh, Hamid R. Shahsavari,
Robert Beitle, and M. Hassan Beyzavi*
Abstract: This study examined the use of nanoparticles created with
recombinant 45-amino acid long peptides fused to green fluorescent
protein (GFPuv) to catalyze twelve representative Suzuki-Miyaura
and Stille coupling reactions. A method was developed to prepare
freeze-dried powders (Pd@GFP) containing protein and synthesized
nanoparticles. Next, coupling reactions were performed in a green
solvent without nanoparticle purification. Pd@GFP had high turnover
frequencies for the synthesis of model compounds including Tykerb®
and could be recycled. This study establishes a potentially cost-
effective approach to prepare heterogeneous catalysts containing
well defined nanoparticles enabling key C–C bond formation leading
to synthetically and pharmaceutically interesting compounds.
Any proposed method for the effective production of
peptides for NP synthesis must recognize the cost-benefit
analysis (CBA) of purification. On the one hand, the temptation to
assume the requirements for highly purified peptide is common in
literature.[3a] We recently demonstrated that even in a highly
impure mixture that includes other biological materials,
nanoparticle formation proceeds through the expected
mechanism of nanoparticle nucleation and growth. A logical
next step for the work is to demonstrate that the NPs formed
under these conditions could also be used without extensive
purification.
[7]
In this work, we describe the successful synthesis of Pd NPs
using three copies of peptide commonly referred to as Pd4
(
TSNAVHPTLRHL) fused to green fluorescent protein (GFPuv).
Inhomogeneous crude Escherichia coli lysate containing (Pd4)
3
-
Palladium (Pd) is a fundamental element of many industrial
GFPuv fusion protein can offer a remarkable scaffold which would
be costly advantageous compared to not only chemically
synthesized peptides, but also organic ligand, metal-coordination
complexes, and organic cages. Furthermore, the successful
synthesis of highly stable and well-dispersed Pd NPs with narrow
processes including H
2
evolution, organic coupling reactions, and
CO
2
reduction.[1] Due to its remarkable catalytic performance,
operational simplicity, and high reaction selectivity, Pd has been
widely used to construct many important molecules applicable in
electronic device development, medicine, and biopharmaceutical
improvement.[2] However, limited resources and high cost of Pd
has encouraged scientists to design a cost-effective route for
fabricating Pd-based catalyst.
3
size distribution using crude lysate containing (Pd4) -GFPuv
fusion protein is described. It is also demonstrated that unpurified
material is capable of controlling size, composition, and
morphology of NPs. We have also investigated the catalytic
One of the most exploited routes is to use Pd nanoparticles
activity of (Pd4)
3
-GFPuv fusion protein-templated Pd NPs
(
Pd NPs). Unique chemical, physical, and mechanical properties
(
Pd@GFP) in Suzuki-Miyaura and Stille coupling reactions. In
of Pd NPs have raised from their high surface area to volume
aspect ratio.[3] Various methods have been developed employing
dendritic architectures and surface capping ligands.[4] On the
other hand, bounding NPs to specific size and shape was
achieved using hollow structures, metal-coordination complexes,
and organic molecular cages.[5] Unfortunately, these methods
were either expensive or unable to eliminate the challenge toward
the size-controllable synthesis of NPs with narrow size
distributions. Biomimetic 3-D ordered structures utilizing short
peptides sequences have been used for NP synthesis to control
size, morphology, and structure.[6] However, developing a cost-
effective approach towards size-controllable NP synthesis
remains unsolved.
addition, the ability of prepared fusion protein-templated Pd NPs
towards anticancer drug (Tykerb®) precursor construction was
studied. The synthesized NPs also showed high performance in
environmentally friendly mixture and their catalytic activities did
not suffer significant loss after cycling test.
(
Pd4)
by overlap extension polymerase chain reaction (SOE PCR)
technique. Using three steps PCR, (Pd4) -GFPuv gene was
3
-GFPuv fusion protein was constructed using splicing
3
synthesized (Figure 1a). The PCR product of the third step and
plasmid were double digested using EcoRI-HF® and NcoI-HF®
restriction enzymes. The final plasmid containing (Pd4) -GFPuv
3
fusion protein codon was obtained after ligation of digested PCR
product and the digested plasmid (Figure 1b). The ligation product
was transformed to into E. coli cells, and the protein mixture was
obtained after the fermentation process.[7]
The NP synthesis process was performed using the protein
extract mixture contained approximately 10% (Pd4) -GFPuv
3
[a]
Dr. I. Mosleh, Prof. R. Beitle; Ralph E. Martin Department of Chemical
Engineering, University of Arkansas, Fayetteville, Arkansas 72701,
USA.
fusion protein (Figure 2a).[7] The TEM image analysis of the
Pd@GFP showed spherical and well-uniformed NPs with an
average size of 2.4 ± 0.7 nm (Figure 2b and c). Prepared particles
I.M. ORCID: 0000-0001-8763-6906
R.B. ORCID: 0000-0001-8859-8358
[b]
Dr. H. R. Shahsavari, Prof. M. H. Beyzavi; Department of
Chemistry and Biochemistry, University of Arkansas, Fayetteville,
Arkansas 72701, United States.
E-mail: beyzavi@uark.edu https://beyzavigroup.uark.edu/
3
using lysate without (Pd4) -GFPuv fusion protein were neither
discrete nor consistent in the absence of Pd4 peptide (Figure S1).
It could be argued that Pd4 sequence is required to obtain uniform
NPs as agglomeration, non-uniformity, and irregularity of particles
occurred due to lack of nucleation site needed to anchor to the
NPs in the absence of Pd4 sequence. It is expected that the
histidine residues in the structure of Pd4 peptide provide suitable
nucleation sites for Pd NPs formation due to the coordination of
M.H.B. ORCID: 0000-0002-6415-8996
[c]
Dr. H. R. Shahsavari; Department of Chemistry, Institute for
Advanced Studies in Basic Sciences (IASBS), Zanjan 45137-66731,
Iran.
H.R.S. ORCID: 0000-0002-2579-2185
On sabbatical leave from IASBS, Zanjan, Iran.
‖
Supporting information for this article is given via a link at the end of
the document.
Pd+2 by the peptide. By adding the NaBH
as a reducing agent,
4
1
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Pd NPs were formed by means of coalescence, Ostwald ripening,
and oriented attachment (OA).[7] The growth of Pd NPs was also
nm. (Pd4)
3
-GFPuv fusion proteins would prevent Pd NPs from
agglomeration through wrapping around the NPs and resulted in
highly stable Pd NPs.
inhibited by histidine residues present in (Pd4)
3
-GFPuv fusion
protein structure resulting in NPs with an average size of 2.4 ± 0.7
Figure 1. Illustration of cloning using PCR and overview of plasmid construction. A synthetic gene was constructed using SOE PCR.
The formation of Pd NPs was also confirmed by energy-
dispersive X-ray spectroscopy (EDX) as shown in Figure 2d.
Based on the representative EDX spectrum of the Pd@GFP, Pd
peaks were detected at 3.3 and 21.2 keV.
would affect salt concentration in the soluble fraction of the lysate,
the presence of NaCl is not surprising. Furthermore, the existence
of O, N, and C elements is attributed to the organic materials and
crude lysate in Pd@GFP structure. As shown in Figure 3, Pd 3d
XPS spectrum shows the characteristic peaks of 3d3/2,5/2 doublets.
The peaks at the binding energy (BE) around 341.0 eV (Pd 3d3/2
)
and 334.5 eV(Pd 3d5/2) are attributed to Pd0 species, and the
peaks around BE of 343.7 eV (Pd 3d3/2) and 337.8 eV(Pd 3d5/2
)
II
are related to Pd species, which probably result from the
0
[8]
reoxidation of Pd . Therefore, the chemical composition of
Pd@GFP as catalyst was confirmed by XPS. The percentage of
Pd was determined by inductively coupled plasma mass
spectroscopy (ICP-MS) to be 1.2% (Figure S3). SEM analysis of
Pd@GFP indicated that Na, Cl, O, and C were also present and
accompanied the Pd NPs (Figure S4).
The C–C coupling reactions, Suzuki-Miyaura and Stille,
provided information on the catalytic activity of Pd@GFP. Based
on optimization (Tables S1-S4), coupling reactions were
-
5
performed using 0.5 mmol% of Pd (5×10 eqiv.) and base (3
equiv.) in a green mixture of EtOH/H O (1:1, v/v) at 80 °C.
2
Different aryl-iodides and -bromides containing electron-rich and
electron-deficient groups reacted satisfactorily with various
phenylboronic acids or phenyltin compounds (Table 1) furnished
high to excellent yields. It was also found that different
Figure 2. (a) Schematic of Pd@GFP, (b) TEM image of Pd@GFP, (c) size
distribution of Pd@GFP, and (d) EDX analysis of Pd@GFP. The detection of
the copper in the EDX is because of the carbon-coated copper (CCC) grid used
for TEM analysis.
2 3 3
substituents such as –CH OH, –CHO, –COCH , –OCF , and –F
remained intact during these transformations. Thus, Pd@GFP
was determined to be an efficient, inexpensive, and green catalyst
for the Suzuki-Miyaura coupling reaction.
X-ray photoelectron spectroscopy (XPS) confirms the
existence of Na, Cl, O, N, Pd, and C elements (Figure S2).
Typically, NaCl concentration in LB is estimated to be within the
range of 5-10 mg/g. Since neither sonication nor centrifugation
In order to extend the potency of our Pd@GFP catalyst, the
synthesis of Tykerb® precursor was performed.[9] Precursor
synthesis of Tykerb® and an anticancer drug requires the Suzuki-
Miyaura reaction to couple 5-formyl-2-furanylboronic acid with
2
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COMMUNICATION
iodoquinazoline (Scheme 1).[9a] By employing Pd@GFP, the
precursor of Tykerb® was obtained in a good yield (73%) which
confirmed that the catalyst is a suitable candidate causing no
conflict with the functional groups present in the drug precursor
Environmental protection concerns as well as cost-effective
approaches for appropriate catalyst used in chemical reactions
are of high importance. The reusability of Pd@GFP catalyst for
Suzuki-Miyaura and Stille couplings was investigated (Figure S5).
The catalyst was reused for five times and the catalytic activity
decrease of about 13% and 18% after five cycles was achieved
in Suzuki-Miyaura and Stille coupling reactions, respectively
(
Scheme 1). This achievement can be considered as a
remarkable applicability of this protocol for the synthesis of
pharmaceutically important complex molecules.
(
Figure S5). Also, no aggregation or morphological changes were
observed in used catalyst after five cycles and the Pd NPs
remained spherical and uniformed without any aggregation. The
average size of nanoparticles remained almost unchanged (2.2 ±
0.7 nm in Suzuki-Miyaura coupling and 2.1 ± 0.6 nm in Stille
coupling) confirming an excellent recyclability (Figure 4). To
confirm the nature of the catalyst, the filtrate of the reaction
mixture showed no progress indicating that the catalyst is indeed
heterogeneous (Figure S6).
Cl
F
I
N
OH
B
HN
O
Cl
O
N
N
N
O
+
HO
O
NH
F
O
O
Scheme 1. Tykerb® precursor route of synthesis, Yield 73%.
Figure 3. Pd3d XPS spectrum of Pd@GFP catalyst.
Table 1. Substrate scope for Suzuki-Miyaura and Stille coupling reactions.a
Yield b (%)
97
TOF(h-1)
12,933
Entry
1
Ar—X
Ar’—R
Time (h)
1.5
2
3
4
2
6
2
86
83
88
8,600
2,766
8,800
5
6
2.5
15
94
82
7,520
1,093
7
8
16
4
69
95
920
4,750
Yield b (%)
96
TOF (h-1)
3,324
Entry
9
Ar—X
Ar’—R
Time (h)
5.75
1
0
1
18
12
72
86
800
1
1,433
3
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12
40
93
465
a
Reaction conditions: Aryl halide (0.1 mmol), arylboronic acid (1.2 mmol), K
2
CO
3
(0.3 mmol) (entries 1-8), and Aryl halide (0.1 mmol), Phenyltin
O:EtOH (1:1, v/v) and refluxed under N
2
compound (1.2 mmol), K
under 80 °C. Yields were determined by HPLC.
3
PO (0.3 mmol) (entries 9-12) and Pd NPs (0.005 µmol) were mixed in 1 mL H
4
2
b
It has been reported that NPs prepared using dendritic
architectures and surface capping ligands may show better
stability, but their catalytic activity is low.[10] This is attributed to
their low surface accessibility while most of the surface is covered
by organic supports. Also, employing Pd@GFP preparation, the
particle size was dictated by the structure of Pd4 due to the
structure of Pd4 peptide fused to GFPuv protein. Regarding to the
catalytic activity, Pd@GFP showed slightly higher catalytic activity
Acknowledgements
This work was supported by the University of Arkansas through
the startup funds to MHB, Arkansas Bioscience Institute and the
Ralph E. Martin Department of Chemical Engineering.
Keywords: Palladium nanoparticles • Peptides • Bio-templated
synthesis • Heterogenous catalysis • Coupling reactions.
3
-1
(
TOF = 3.3 × 10 h ) comparing to the Pd NPs prepared using
chemically synthesized Pd4 peptides.[3a, 11] This shows that the
surface accessibility and catalytic activity of Pd NPs prepared
through recombinant approach were not compromised. In other
words, a recombinant approach to provide Pd4 or a similar
construct appears to be attractive from both a scientific and
economic standpoint.
To conclude, a facile aqueous solution phase NP synthesis
method was exploited in a heterogeneous biological protein
environment. Pd@GFP catalyst was utilized for Suzuki-Miyaura
and Stille coupling reactions in green solvents. The catalyst was
more active compared to chemically synthesized peptide-
templated Pd NPs as exhibits higher catalytic activity with very
good stability (5 cycles). Also, Pd@GFP was successfully used
for the preparation of an anti-cancer drug (Tykerb®) precursor.
Pd@GFP not only extends the application of peptide-templated
NPs, but also provides a cost-effective approach to design
heterogeneous catalysts. This catalyst resulted in a three order of
magnitude decrease in cost due to elimination of purification steps.
[
1]
a) W. Fu, Y. Cao, Q. Feng, W. R. Smith, P. Dong, M. Ye, J. Shen,
Nanoscale 2019, 11, 1379-1385; b) H. Huang, H. Jia, Z. Liu, P. Gao, J.
Zhao, Z. Luo, J. Yang, J. Zeng, Angew. Chem. Int. Ed. 2017, 56, 3594-
3598; c) X. Liu, S. Gao, P. Yang, B. Wang, J. Z. Ou, Z. Liu, Y. Wang,
Appl. Mater. Today 2018, 13, 158-165.
[2]
3]
a) I. P. Beletskaya, F. Alonso, V. Tyurin, Coord. Chem. Rev. 2019, 385,
137-173; b) L. C. Campeau, N. Hazari, Organometallics 2019, 38, 3-35.
[
a) N. M. Bedford, H. Ramezani-Dakhel, J. M. Slocik, B. D. Briggs, Y. Ren,
A. I. Frenkel, V. Petkov, H. Heinz, R. R. Naik, M. R. Knecht, ACS Nano
2015, 9, 5082-5092; b) Y. Xia, K. D. Gilroy, H. C. Peng, X. Xia, Angew.
Chem. Int. Ed. 2017, 56, 60-95; c) B. Karimi, H. Barzegar, H. Vali, Chem.
Commun. 2018, 54, 7155-7158; d) W. Wang, C. F. Anderson, Z. Wang,
W. Wu, H. Cui, C. J. Liu, Chem. Sci. 2017, 8, 3310-3324.
[4]
a) X. Liu, D. Gregurec, J. Irigoyen, A. Martinez, S. Moya, R. Ciganda, P.
Hermange, J. Ruiz, D. Astruc, Nat. Commun. 2016, 7, 13152; b) A.
Heuer-Jungemann, N. Feliu, I. Bakaimi, M. Hamaly, A. Alkilany, I.
Chakraborty, A. Masood, M. F. Casula, A. Kostopoulou, E. Oh, K.
Susumu, M. H. Stewart, I. L. Medintz, E. Stratakis, W. J. Parak, A. G.
Kanaras, Chem. Rev. 2019, 119, 4819-4880.
[
[
5]
6]
a) B. Li, N. You, Y. Liang, Q. Zhang, W. Zhang, M. Chen, X. Pang, Energy
Environ. Mater. 2019, 2, 38-54; b) N. B. Debata, D. Tripathy, H. S. Sahoo,
Coord. Chem. Rev. 2019, 387, 273-298; c) L. Qiu, R. McCaffrey, Y. Jin,
Y. Gong, Y. Hu, H. Sun, W. Park, W. Zhang, Chem. Sci. 2018, 9, 676-
680.
a) N. M. Bedford, A. R. Showalter, T. J. Woehl, Z. E. Hughes, S. Lee, B.
Reinhart, S. P. Ertem, E. B. Coughlin, Y. Ren, T. R. Walsh, B. A. Bunker,
ACS Nano 2016, 10, 8645-8659; b) C. J. Munro, M. R. Knecht, Curr.
Opin. Green Sustain. Chem. 2018, 12, 63-68; c) T. R. Walsh, M. R.
Knecht, Chem. Rev. 2017, 117, 12641-12704; d) Y. Cheng, J. Zheng, C.
Tian, Y. He, C. Zhang, Q. Tan, G. An, G. Li, Asian J. Org. Chem. 2019,
8, 526-531.
[
7]
a) I. Mosleh, M. Benamara, L. Greenlee, M. H. Beyzavi, R. Beitle, Mater.
Lett. 2019, 252, 68-71; b) R. Tejada-Vaprio, I. Mosleh, Mosleh, R. P.
Mukherjee, H, Aljewari, M. Fruchtl, A. Elmasheiti, N. Bedford, L.
Greenlee, M. H. Beyzavi, R. Beitle, Biotechnol Prog. 2020, 36, e2956.
D. Han, Z. Bao, H. Xing, Y. Yang, Q. Ren, Z. Zhang, Nanoscale 2017, 9,
[
[
8]
9]
6026-6032.
a) S. Mahboobi, A. Sellmer, M. Winkler, E. Eichhorn, H. Pongratz, T.
Ciossek, T. Baer, T. Maier, T. Beckers, J. Med. Chem. 2010, 53, 8546-
8555; b) G. Erickson, J. Guo, M. McClure, M. Mitchell, M.-C. Salaun, A.
Whitehead, Tetrahedron Lett. 2014, 55, 6007-6010.
Figure 4. (a) TEM image, and (b) particle size distribution of Pd@GFP catalyst
after five cycles for Suzuki-Miyaura coupling. (c) TEM image, and (d) particle
size distribution of Pd@GFP catalyst after five cycles for Stille coupling.
[10] a) B. A. Rozenberg, R. Tenne, Prog. Polym. Sci. 2008, 33, 40-112; b) S.
Chatterjee, S. K. Bhattacharya, ACS Omega 2018, 3, 12905-12913; c)
A. K. Diallo, C. Ornelas, L. Salmon, J. R. Aranzaes, D. Astruc, Angew.
Chem. Int. Ed. 2007, 46, 8644-8648.
[
11] R. Coppage, J. M. Slocik, H. Ramezani-Dakhel, N. M. Bedford, H. Heinz,
Supporting Information (see footnote on the first page of this article):
R. R. Naik, M. R. Knecht, J. Am. Chem. Soc. 2013, 135, 11048-11054.
.
Full experimental details and characterization data (NMR and
Mass spectra) for compounds.
4
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Entry for the Table of Contents
COMMUNICATION
Imann Mosleh, Hamid R. Shahsavari,
Robert Beitle, and M. Hassan Beyzavi*
Page No. – Page No.
Recombinant Peptide Fusion Protein-
templated Palladium Nanoparticles for
Suzuki-Miyaura and Stille Coupling
Reactions
A peptide-templated Palladium nanoparticles are synthesized and utilized for
Suzuki-Miyaura and Stille coupling reactions.
5
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